Giuseppe Bellavia

Protein Thermal Denaturation and Matrix Glass Transition in Different Protein−Trehalose−Water Systems

Biopreservation by saccharides is a widely studied issue due to its scientific and technological importance; in particular, ternary amorphous protein-saccharide-water systems are extensively exploited to model the characteristics of the in vivo biopreservation process. We present here a differential scanning calorimetry (DSC) study on amorphous trehalose-water systems with embedded different proteins (myoglobin, lysozyme, BSA, hemoglobin), which differ for charge, surface, and volume properties. In our study, the protein/trehalose molar ratio is kept constant at 1/40, while the water/sugar molar ratio is varied between 2 and 300; results are compared with those obtained for binary trehalose-water systems. DSC upscans offer the possibility of investigating, in the same measurement, the thermodynamic properties of the matrix (glass transition, T(g)) and the functional properties of the encapsulated protein (thermal denaturation, T(den)). At high-to-intermediate hydration, the presence of the proteins increases the glass transition temperature of the encapsulating matrix. The effect mainly depends on size properties, and it can be ascribed to confinement exerted by the protein on the trehalose-water solvent. Conversely, at low hydration, lower T(g) values are measured in the presence of proteins: the lack of water promotes sugar-protein interactions, thus weakening the confinement effect and softening the matrix with respect to the binary system. A parallel T(den) increase is also observed; remarkably, this stabilization can reach ∼70 K at low hydration, a finding potentially of high biotechnological relevance. A linear relationship between T(g) and T(den) is also observed, in line with previous results; this finding suggests that collective water-trehalose interactions, responsible for the glass transition, also influence the protein denaturation.

Thermal denaturation of myoglobin in water--disaccharide matrixes: relation with the glass transition of the system.

Proteins embedded in glassy saccharide systems are protected against adverse environmental conditions [Crowe et al. Annu. Rev. Physiol. 1998, 60, 73-103]. To further characterize this process, we studied the relationship between the glass transition temperature of the protein-containing saccharide system (T(g)) and the temperature of thermal denaturation of the embedded protein (T(den)). To this end, we studied by differential scanning calorimetry the thermal denaturation of ferric myoglobin in water/disaccharide mixtures containing nonreducing (trehalose, sucrose) or reducing (maltose, lactose) disaccharides. All the samples studied are, at room temperature, liquid systems whose viscosity varies from very low to very large values, depending on the water content. At a high water/saccharide mole ratio, homogeneous glass formation does not occur; regions of glass form, whose T(g) does not vary by varying the saccharide content, and the disaccharide barely affects the myoglobin denaturation temperature. At a suitably low water/saccharide mole ratio, by lowering the temperature, the systems undergo transition to the glassy state whose T(g) is determined by the water content; the Gordon-Taylor relationship between T(g) and the water/disaccharide mole ratio is obeyed; and T(den) increases by decreasing the hydration regardless of the disaccharide, such effect being entropy-driven. The presence of the protein was found to lower the T(g). Furthermore, for nonreducing disaccharides, plots of T(den) vs T(g) give linear correlations, whereas for reducing disaccharides, data exhibit an erratic behavior below a critical water/disaccharide ratio. We ascribe this behavior to the likelihood that in the latter samples, proteins have undergone Maillard reaction before thermal denaturation.

Proteins in amorphous saccharide matrices: Structural and dynamical insights on bioprotection

Bioprotection by sugars, and in particular trehalose peculiarity, is a relevant topic due to the implications in several fields. The underlying mechanisms are not yet clearly elucidated, and remain the focus of current investigations. Here we revisit data obtained at our lab on binary sugar/water and ternary protein/sugar/water systems, in wide ranges of water content and temperature, in the light of the current literature. The data here discussed come from complementary techniques (Infrared Spectroscopy, Molecular Dynamics simulations, Small Angle X-ray Scattering and Calorimetry), which provided a consistent description of the bioprotection by sugars from the atomistic to the macroscopic level. We present a picture, which suggests that protein bioprotection can be explained in terms of a strong coupling of the biomolecule surface to the matrix via extended hydrogen-bond networks, whose properties are defined by all components of the systems, and are strongly dependent on water content. Furthermore, the data show how carbohydrates having similar hydrogen-bonding capabilities exhibit different efficiency in preserving biostructures.Graphical abstract

How Does Glycerol Enhance the Bioprotective Properties of Trehalose? Insight from Protein–Solvent Dynamics

We present Raman investigations on lysozyme/trehalose/glycerol solutions at low water content, from room temperature up to the occurrence of the protein thermal denaturation. We studied the Amide I band and the low-frequency spectrum as a function of the glycerol content. The former allows us to monitor the protein unfolding; the latter probes the protein and solvent dynamics in anharmonic and quasi-harmonic regimes. It was shown that adding a small amount of glycerol to trehalose stiffens the dry matrix in which proteins are embedded, thus improving their stability. The analysis of the Amide I band reveals that glycerol enhances the stabilization effect of trehalose on proteins for low water content, but still liquid, systems. Data show that the protein unfolding temperature has a maximum value around 5% Glyc/TRE g/g. The overlapping low-frequency contributions, corresponding to fast anharmonic and quasi-harmonic motions, respectively, related to the mean square displacement ⟨u(2)⟩ and the vibrational density of states (VDOS) usually determined by neutron scattering experiments, have been carefully analyzed to understand the effect of glycerol. The intensity of the quasi-elastic scattering (QES) peak reveals a dynamical-like transition at high temperatures, close to the denaturation temperature. This one, as well as the low-frequency vibrational modes, reflects the same enhanced trend of the Amide I band with respect to the glycerol concentration, but at lower temperatures. A linear correlation is found among the transition temperatures of both the dynamical-like transition and the low-frequency modes, as well as the temperature dependent change of the Amide I frequency. This confirms the solvent dynamics as a necessary precursor to promote protein unfolding. Glycerol anti-plasticizes the matrix with respect to the trehalose by enhancing the stability of the protein in a more rigid trehalose/water/glycerol matrix. As expected from the analysis of the Amide I band, the maximum effect of glycerol on trehalose is determined for 5% Glyc/TRE content.